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Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Biological Procedures Online
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275047/ https://www.ncbi.nlm.nih.gov/pubmed/18385809 http://dx.doi.org/10.1251/bpo135 |
Sumario: | B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x10(6) purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x10(6 )primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population. |
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